Chromatographic methods are very useful in the detection, identification and quantitation of toxicologically relevant substances. If retention in a chromatography system will be used as an identification parameter, a way to express the chromatographic behavior of each substance is needed. This must be a unique value, as independent as possible of all the chromatography conditions, except the nature of the stationary phase and the substance itself. Retention indices fulfill these conditions. We describe the equations, software and chromatographic conditions used in the calculations of Retention Indices based in the use of drugs as a series of standards and the inter and intralaboratory reproductibility. Using drugs as reference standards, an intra laboratory dispersion within a range of +/- 2 RI units with a standard deviation of one RI units is achieved. The dispersion of the differences of RI in literature (calculated with n-alkanes as standards and in both packed an capillary columns) and the "in home" values are in the range of +/- 25 RI units with a standard deviation of 8.1 units. We finally describe the application of Retention Indices in Forensic Toxicology, specially in the screening of the "general unknown" based on the premises of the "Systematic Toxicological Analysis" and in the development and validation of quantitative techniques.